title: Development of a bifunctional xylosidase/arabinosidase gene as a reporter gene for the gram-negative anaerobes Bacteroides and Porphyromonas, and Escherichia coli.
abstract: Members of the genera Bacteroides and Porphyromonas are common inhabitants of the human intestinal and oral microflora, and certain species are capable of causing disease states in humans. Genetic studies of these organisms are important for determining factors involved in the development of such diseases. A reporter gene for transcriptional fusions may prove useful for studies of gene regulation in these organisms. Bacteroides ovatus is a normal inhabitant of the human intestinal tract and is one of the few Bacteroides species capable of degrading xylan, a major component of fiber in the diet. A gene encoding for a bifunctional xylosidase/arabinosidase (XA) enzyme was previously cloned in our laboratory from B. ovatus V975 as part of a xylan-inducible operon. The XA gene was isolated by polymerase chain reaction and subcloned into the E. coli plasmid pBluescript II KS+. The XA gene is under transcriptional regulation in E. coli by the lac promoter, and both activities can be induced with isopropylthio-beta-galactoside (IPTG). The XA gene was subcloned into E. coli/Bacteroides shuttle vectors and introduced by conjugation into different Bacteroides species and Porphyromonas gingivalis. The results of transcriptional fusions in Bacteroides species and E. coli were evaluated. The characteristics of the XA reporter system are the low background or total lack of arabinosidase and xylosidase activities in most Bacteroides species, P. gingivalis, and E. coli, and the ease of enzymatic assays. In addition, bacterial colonies can be screened directly on agar plates by fluorescence with methylumbelliferyl derivatives as substrates for either enzymatic activity.
authors.affiliation: Fermentation Biochemistry Research Unit, National Center for Agricultural Utilization Research, USDA, Peoria, IL 61604, USA.
authors.forename: T R
authors.initials: TR
authors.lastname: Whitehead
mesh_terms: D019677:Artificial Gene Fusion; D001426:Bacterial Proteins / Q000032:analysis; D001439:Bacteroides / Q000201:enzymology / Q000235:genetics*; D003001:Cloning, Molecular; D003227:Conjugation, Genetic; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D015971:Gene Expression Regulation, Enzymologic; D017930:Genes, Reporter; D006026:Glycoside Hydrolases / Q000235:genetics* / Q000378:metabolism; D007544:Isopropyl Thiogalactoside / Q000378:metabolism; D007763:Lac Operon; D010957:Plasmids; D016133:Polymerase Chain Reaction; D016966:Porphyromonas gingivalis / Q000235:genetics* / Q000378:metabolism; D011401:Promoter Regions, Genetic; D015183:Restriction Mapping; D014995:Xylosidases / Q000235:genetics* / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D007544:Isopropyl Thiogalactoside; D006026:Glycoside Hydrolases; D014995:Xylosidases; C019713:alpha-N-arabinofuranosidase